Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues. The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield, An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity. Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle. However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wildtype virus. The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications. In this work, the dependence of the vector yield on the genetic background of the virus is examined. In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system. After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost.